We have determined the full-length ClpA structure, which was the first type-2 AAA protein. ClpA, an Hsp100/Clp chaperone and an integral component of the ATP-dependent ClpAP protease, participates in regulatory protein degradation and the dissolution and degradation of protein aggregates. The crystal structure of the ClpA subunit reveals an N-terminal domain with pseudo two-fold symmetry and two AAA+ modules (D1 and D2) each consisting of a large and a small sub-domain with ADP bound in the sub-domain junction. The N-domain interacts with the D1 domain in a manner similar to adaptor-binding domains of other AAA+ proteins. D1 and D2 are connected head-to-tail consistent with a cooperative and vectorial translocation of protein substrates. In a planar hexamer model of ClpA, built by assembling ClpA D1 and D2 into homohexameric rings of known structures of AAA+ modules, the differences in D1-D1 and D2-D2 interfaces correlate with their respective contributions to hexamer stability and ATPase activity. We also determined the complex structure of ClpA with its regulator protein ClpS that involves in substrate selection. Substrate selectivity and proteolytic activity for the E. coli ATP-dependent protease, ClpAP, is modulated by an adaptor protein, ClpS. ClpS binds to ClpA, the regulatory component of the ClpAP complex. We report the crystal structure of ClpS in complex with the isolated N-terminal domain of ClpA in two different crystal forms at 2.3 A and 3.3 A resolution. The ClpS structure forms a alfa/beta-sandwich and is topologically analogous to the C-terminal domain of the ribosomal protein L7/L12. ClpS contacts two surfaces on the N-terminal domain in both crystal forms; the more extensive interface was shown to be favored in solution by protease protection experiments. The N-terminal 20 residues of ClpS are not visible in crystal; removal of the first 17 residues produces ClpS/N, which binds to the ClpA N-domain but no longer inhibits ClpA activity. A zinc-binding site involving two His and one Glu residue was identified crystallographically in the N-terminal domain of ClpA. In a model of ClpS bound to hexameric ClpA, ClpS is oriented with its N-terminus directed toward the distal surface of ClpA, suggesting that the N-terminal region of ClpS may affect productive substrate interactions at the apical surface or substrate entry into the ClpA translocation channel.Our recent work has been focusing on the human AAA protein p97. Mutations in p97, the major cytosolic AAA chaperone, cause inclusion body myopathy associated with Pagets disease of the bone and frontotemporal dementia (IBMPFD). IBMPFD mutants have single amino acid substitutions at the interface between the N-terminal domain (N-domain) and the adjacent AAA domain (D1), resulting in a reduced affinity for ADP. The structures of p97 N-D1 fragments bearing IBMPFD mutations adopt an atypical N-domain conformation in the presence of Mg2+-ATPgS, which is reversible by ADP, demonstrating for the first time the nucleotide-dependent conformational change of the N-domain. The transition from the ADP- to the ATPgS-bound state is accompanied by a loop-to-helix conversion in the N-D1 linker and by an apparent re-ordering in the N-terminal region of p97. X-ray scattering experiments suggest that wild type p97 subunits undergo a similar nucleotide dependent N-domain conformational change. We propose that IBMPFD mutations, by destabilizing the ADP bound form, alter the timing of the transition between nucleotide states and consequently interfere with the interactions between the N-domains and their substrates.Wild type and mutant N-D1 fragments were also studied in the presence of ATPgS or ADP by SAXS. The radii of gyration (Rg) are consistently 3-5 A smaller for the ATPgS-bound N-D1 fragment as compared to the ADP-bound form. The conformational change of N-D1 in solution can also be demonstrated by the distance distribution functions, p(r), in which a significant shift in the distribution towards shorter vectors was observed for the ATPgS-bound N-D1 fragments, This shift in p(r) is most obvious at vector lengths beyond 90 A, consistent with the large-scale N-domain conformational change. Furthermore, calculated changes in the distribution function based on crystal structures are in agreement with the experimentally obtained distribution functions, suggesting that the crystallographically observed differences in conformation of the N-domain exist in solution not only for p97 mutants but also for wild type p97. Using isothermal titration calorimetry (ITC), we determined a Kd value of 0.88 uM towards ADP for the wild type N-D1 with a stoichiometry of 0.35, suggesting only 2 out of 6 sites are available for binding, which is consistent with previously reported values. By contrast, mutant p97 N-D1 fragments displayed reduced binding affinities for ADP and the level of reduction is site dependent. For example, the R155H mutant showed a maximum reduction with a Kd of 4.25 uM. Notably, the changes in the binding stoichiometry are correlated with the changes in binding affinities for the mutants. Consistent with the previous findings, wild type p97 showed a Kd value for ATPgS of 0.89 uM, similar to that for ADP. Unexpectedly, the titration profiles with ATPgS for mutants were biphasic and can only be fitted to a two-site model. The Kd values for the high affinity site were well determined and close to 0.1 uM for all mutants, whereas those for the low affinity site were associated with significant errors. Again, mutant p97 displayed higher stoichiometry than wild type in the ATPgS titration experiments.A model with four nucleotide-binding states for the ATP cycle in the D1-domain was proposed. First, there is an ATP state, with ATP bound and the N-domain in the Up-conformation. In a wild type p97 hexamer, due to non-exchangeable, pre-bound ADP, not all subunits will have their N-domains in the Up-conformation even with an excess amount of ATP in solution. We therefore hypothesize that there is an ADP-locked state, with non-exchangeable, pre-bound ADP at the D1 site and the N-domain in the Down-conformation. This state appears to be important for wild type p97 function and the pre-bound ADP is particularly difficult to exchange. The structure of the N-D1 fragment of wild type p97 may represent this conformation. In a third state, termed ADP-open, ADP is bound but exchangeable. This state was observed for mutant p97 by its biphasic ITC titration profile and is presumably in equilibration with the ADP-locked state. The structure of R155H with bound ADP represents this conformation. The fourth state is the Empty state, with nucleotide-binding sites unoccupied and the N-domain in an unknown position. The difference between the wild type and mutants, however, lies in the transition between the ADP-locked state and the ADP-open state. We propose that in the wild type protein this transition is tightly controlled and characterized by the asymmetry in nucleotide binding states in D1-domains of different subunits, resulting in a low concentration of the ADP-open state, whereas in IBMPFD mutants, this control mechanism is altered, leading to a high concentration of subunits in the ADP-open state.